PTH is the primary regulator of calcium homeostasis and bone metabolism in mammals. Its signaling system has served as a major target for the development of novel anabolic therapeutic approaches for osteoporosis. However, the exact mechanisms by which PTH exerts its actions in bone are not fully understood. Significant progress has been made in determining the mechanisms of PTH downstream signaling, but the detailed downstream signaling mechanisms do not seem to provide an explanation for the anabolic effects of PTH on bone. Endocytosis of seven-transmembrane receptors, including PTH1R, coordinates different signals and acts as a fundamental organizer of the cell, controlling many cellular processes, including cell fate determination. Thus, we began to consider whether PTH-induced endocycytosis modulates and integrates other anabolic signals. Our preliminary data show that PTH induces recruitment of the TGF type II receptor (TRII) to PTH1R, thereby facilitating their signaling, and that both receptors are internalized as a TRII/PTH1R complex. Immunoprecipitation and FRET experiments demonstrated formation of a triple complex consisting of TRII, PTH1R, and PTH (1-84). As a result, signaling of TGF and PTH is coordinated. Expression of TRII enhances PTH-induced endocytosis of PTH1R and reduces the amount of cell surface PTH1R. Consequently, cAMP production, PKC and ERK1/2 activities are down-regulated. Conversely, the recruitment of TRII to PTH1R by PTH resulting in under internalization dampened TGF-induced Smad signaling. Therefore, we hypothesize that PTH-induced endocytosis of TRII and PTH1R as a complex integrates the signals transduced from both TGF and PTH. Thus, the different effects of PTH on bone are specified by the context PTH and TGF signals and the generation of coordinated signals. In this way, while PTH elicits downstream signaling, it also can modulate differentiation, proliferation and coupling of osteoprogenitors through induction of endocytosis of TRII. The proposal is organized into three aims: analysis of the signaling mechanisms in vitro, and in TRII and TGF1 knockout mice. In Aim I, the effects of PTH- induced endocytosis of the TRII/PTH1R complex on cellular signaling will be examined. The phosphorylation sites of PTH1R cytoplasmic domain by TRII will be characterized. In Aim II, the effects of PTH-induced TRII endocytosis on bone remodeling will be characterized in both TRII conditional knockout mice. The role of TGF1 in coupling bone resorption and formation in PTH-induced anabolic bone formation will be examined in TGF1 knockout mice in the Aim III. PUBLIC HEALTH RELEVANCE: PTH(1-34), the N-terminal synthetic peptide of PTH, is used clinically and represents the only anabolic agent that is available for treatment of patients with osteoporosis. PTH ligands and their signaling components provide potential major targets for the development of novel anabolic therapeutic approaches for osteoporosis. However, the current state of knowledge of the downstream signaling mechanisms does not provide an adequate explanation for the anabolic effects of PTH on bone. Here, we propose a paradigm shift in the approach to this problem. Based on our preliminary data, we hypothesize that PTH regulates other anabolic signals, such as TGF, and coordinates the signaling processes. We have found that PTH down-regulates TGF signaling in modulating osteoblast differentiation for bone formation. Validation of our findings could account for the paradoxical effects of PTH on bone: a catabolic effect on continuous administration of PTH and an anabolic effect on intermittent administration. Further elucidation of the hypothetical mechanisms, as proposed herein, could result in the development of novel anabolic therapies.